Characterizing Cre/Lox

Measuring Gene Expression

iGEM Davidson – Missouri Western 2010:Fundamental Advancements in Biology and the Knapsack Problem

The Davidson/Missouri Western multidisciplinary team is using synthetic biology to address a mathematical problem in ''Escherichia coli''. Specifically, we are addressing the Knapsack Problem, an NP-complete problem that asks, “Given a finite number of weighted items, can one find a subset of these items that completely fills a knapsack of fixed capacity?”

In our design, weighted items are represented by versions of ''TetA'' genes that confer measurably distinct levels of tetracycline resistance. We have altered the codons of the wild type ''TetA'' gene, optimizing and de-optimizing several segments of the coding sequence. Each ''TetA'' variant is coupled with a distinctive fluorescent gene, and each pair of genes is flanked by ''lox'' sites. In the presence of Cre protein, the ''lox'' mechanism either inverts or excises the coding sequence, yielding different combinations of expressed ''TetA'' variants. An expressed variant corresponds to an item being placed in the knapsack. Over-expression of ''TetA'' results in cell death, which represents exceeding the capacity of the knapsack. Under-expression of ''TetA'' causes the cells to stop growing due to tetracycline in the growth medium, which represents not completely filling the knapsack. Surviving cells correspond to cells within a certain range of ''TetA'' production and the fluorescence tag allows for comparative measurement within this range.

The team is also working to develop software tools relevant to the specific project and applicable to projects in the wider synthetic biology community.
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